The alarming persistence of Legionella species in treated water systems has led scientists to investigate the extent of their distribution and possible causes for their survival from disinfection treatment. In the past few years, studies have shown that Legionella can multiply intracellularly within protozoa. The purpose of the proposed study is to examine possible persistence of Legionellae caused by association with cyst-forming protozoa. Specifically the objectives are: 1) To assess the role of protozoan cysts in enhancing survival of Legionella; 2) to test the effect of biocides on enhancement of cyst formation and consequent entrapment of legionellae; and 3) to screen environments such as drinking water storage tanks and air-conditioning cooling towers for evidence of legionellae within protozoan cysts. Cyst-forming protozoa will be allowed to ingest Legionella and encyst. Cysts will be chemically treated to kill the bacteria on the outside but allow protozoa to excyst, after which they will be lysed and examined for recovery of viable Legionella. Age of cysts up to 2 years will be tested. Resistance of Legionella within cysts to chlorine and chloramine will be assessed. Polymerase chain reaction (PCR) for amplification of Legionella genes, followed by detection with a gene probe, will be employed for samples which are negative for growth on BCYE agar or by fluorescent antibody testing. Cysts may be a mechanism of not only survival of the bacterial pathogen, but also of transmission. These studies may help lead to more effective disinfection strategies for controlling Legionella species in treated drinking water.